PROJECT SUMMARY V-ATPases are highly conserved, multisubunit proton pumps that acidify organelles including lysosomes, endosomes, Golgi apparatus, and secretory granules in all eukaryotic cells. Each of these organelles requires tuning of its luminal pH to a narrow range for its function. However, it is not fully understood how these pH ranges are maintained or how V-ATPases in different organelles respond when environmental conditions challenge local pH gradients. Our central hypothesis is that PI lipids provide organelle-specific inputs to locally regulate V-ATPase activity, and thus can impact individual subsets of the many functions dependent on V-ATPase activity and organelle acidification. In Aim 1, we use the well- characterized yeast V-ATPase system to characterize binding of the cytosolic domains of the membrane- bound a-subunit isoforms, Vph1 and Stv1, to phosphoinositide (PI) lipids. We have found that vacuole-resident Vph1 is able to bind the vacuolar signaling lipid PI(3,5)P2 and Golgi-resident Stv1 prefers the Golgi-enriched lipid PI(4)P. We will quantify and characterize these binding preferences and probe how binding of specific lipids activates V-ATPases containing individual a-subunit isoforms. Based on recent structures and homology modeling, we will then develop lipid-binding mutants for each isoform. Finally, we will evaluate PI binding specificities of the four human a-subunit isoforms. We have tested three of the four human isoforms and found evidence of PI-specific interactions in vitro. We will characterize specificity and affinities of PI binding and identify potential binding sites. In Aim 2, we address the spatial and temporal consequences of PI lipid binding to intact V-ATPases containing full-length Vph1 and Stv1. We hypothesize that PI lipids exert spatial control by activating V-ATPases in their organelles of residence. Specifically, we propose that: 1) PI(3,5)P2 binding to Vph1-containing V-ATPases induces a conformation that stabilizes interactions between the peripheral V1 sector and the integral membrane Vo sector of the V-ATPase, increasing activity, 2) PI(4)P binding to Stv1- containing V-ATPases promotes retention in the Golgi apparatus, and 3) PI(3)P promotes acidification of the endocytic pathway by interacting with Vph1-containing V-ATPases. In contrast, hyperosmotic stress promotes a rapid and transient rise in PI(3,5)P2 that reversibly activates Vph1-containing V-ATPases in the vacuole. We will analyze the mechanisms of this temporal V-ATPase activation and its role in providing short-term adaptation to salt stress. These experiments link PI and V-ATPase isoform content, two fundamental features of organelle identity. They promise to provide novel insights into the many diseases linked to defective organelle acidification, including neurodegeneration, cancer, and mycobacterial infection, and could suggest new therapeutic routes.